Triple negative breast cancer (TNBC) is a form of breast cancer for which no molecularly targeted therapies have reached clinical approval, leaving few treatment options for TNBC patients. The observation of high RAF/MEK/ERK activity in many TNBC patient samples has prompted the investigation of using MEK1/2 inhibitors as a potential therapeutic strategy. Treating TNBC cell lines and mouse models with the MEK1/2 inhibitor AZD6244 provokes a kinome reprogramming event where the activity and expression of growth- promoting receptor tyrosine kinases, such as PDGFRb, are induced to offset MEK1/2 inhibition and reactivate ERK1/2. Targeting AZD6244-responsive kinases with small molecule inhibitors or siRNA not only attenuates the reactivation of ERK1/2, but also synergizes with AZD6244 to inhibit proliferation and induce apoptosis of TNBC cell lines and cause tumor regression of a genetically engineered mouse model (C3-Tag), suggesting that kinome reprogramming contributes to drug resistance in these models. Understanding the mechanisms of AZD6244-induced kinome reprogramming will inform future studies on the use of MEK1/2 inhibitors, alone or in combination, for treating TNBC. With changes in kinase and cytokine expression, kinome reprogramming caused by AZD6244 treatment is initiated by disrupted regulation of transcription factors governing kinase and cytokine gene expression. The transcription factor c-Myc, which is stabilized by ERK1/2-mediated phosphorylation, was shown to be rapidly degraded in TNBC cells following treatment with AZD6244. Aim 1 will determine how AZD6244-induced degradation of c-Myc, a known transcriptional regulator of PDGFRb, contributes to the enhancement of PDGFRb expression and activity in AZD6244-treated cells. Inducible knockdown of c-Myc or proteasomal inhibition will be applied to resolve the role of c-Myc stability in the AZD6244-induced PDGFRb response. Aim 2 will define the mechanism of ERK1/2 reactivation in the presence of AZD6244. Kinase assays with active and inactive MEK1/2, selective knockdown of MEK isoforms, and quantitative proteomics analysis of RAF/MEK/ERK pathway phosphorylation will be performed to focus specifically on how MEK1/2 can escape inhibition by AZD6244 to reactivate ERK1/2. Taken together, these aims will define how transcription factors regulated by ERK1/2 signaling mediate kinome reprogramming in response to AZD6244, and how the reprogramming events subsequently allow TNBC cells to overcome MEK1/2 inhibition. PUBLIC HEALTH RELEVANCE: Triple negative breast cancer patients have a poor prognosis and limited treatment options. The RAF/MEK/ERK pathway is activated in many triple negative breast cancer patients, and use of MEK1/2 inhibitors is an attractive new strategy for treating these patients. This proposal aims to define how MEK1/2 inhibitors may provoke kinome reprogramming and drug resistance in triple negative breast cancer cells, and will contribute to the development of MEK1/2 inhibitors, alone or in combination with other therapeutics, for the treatment of triple negative breast cancer.